DESCRIPTION: For appropriate gene regulation, sequence specific DNA binding proteins must bind their target sites with high affinity and specificity. Only when appropriately configured can the protein-DNA complex influence the activity of the cell's basal transcription machinery. The studies proposed in this application are aimed at understanding the mechanisms by which protein an DNA sequence influence the dynamic, cooperative structural adjustments in both protein and DNA that lead to variations in the affinity, specificity, and transcriptional regulatory activity of protein-DNA complexes. These studies will investigate the DNA binding and transcriptional regulatory properties of 434 and P22 bacteriophage repressors. This set of proteins forms an excellent model system in which functional, biochemical, and structural data can be compared. The functional significance of sequence-dependent differences in DNA twist and twisting flexibility will be determined from binding studies. The mechanism by which DNA sequence affects repressor-catalyzed transcriptional activation will be inferred from kinetic studies. A detailed view of the sequence determinants of DNA dynamics will be obtained from footprinting investigations, NMR studies of unbound and repressor-bound DNA and binding studies using modified nucleotides. To begin to understand how the structure of 434 repressor contributes to its DNA binding and gene regulatory functions investigators will use both random and site-directed mutagenesis techniques to better define the dimer interface of 434 repressor. The ability of the dimer mutant repressors to distinguish between changes in contacted and non-contacted bases will be determined. Ring closure and DNA bending studies will assess the ability of dimer mutant repressors to both affect and recognize changes in DNA structure. The effects of dimer mutations on repressor-operator complex structure will be determined from binding studies and analysis of chemical and enzymatic footprint patterns Kinetic studies will be performed to investigate the role of protein structure on mutant-repressor catalyzed transcription initiation. A kinetic approach will be taken in an effort to ascertain the role of repressor structural changes in enabling repressor to distinguish between specific and non specific DNA or between various specific sites. These studies will also allow to determine the oligomeric form of repressor that searches th DNA for a specific site.